Phospholipid derivative

ABSTRACT

A phospholipid derivative represented by the formula (1) (Z represents a residue of a compound having 3 to 10 hydroxyl groups; AO represents an oxyalkylene group having 2 to 4 carbon atoms; R 1 CO and R 2 CO represent an acyl group having 8 to 22 carbon atoms; X represents hydrogen atom, an alkali metal atom, ammonium or an organic ammonium; “a” represents an integer of 0 to 4; “b” represents 0 or 1; Q represents hydrogen atom or methyl group; m represents an average number of moles of the oxyalkylene group added; and m, k1, k2, and k3 are numbers satisfying the following conditions: 3≦m≦200, 9≦m×(k1+k2+k3)≦1000, 1≦k1≦2, 0≦k2≦9 and 0≦k3≦9, and 3≦k1+k2+k3≦10), which is highly safe for living bodies, and is suitably used for solubilization and dispersion of physiologically active substances and the like, or in the fields of drug delivery systems such as liposomes and cosmetics.

TECHNICAL FIELD

The present invention relates to a phospholipid derivative containing amulti-arm polyalkylene oxide and a method for producing the same. Thepresent invention also relates to a surfactant, solubilizer, dispersingagent for cosmetics, and lipid membrane structure containing thephospholipid derivative.

BACKGROUND ART

It is known that microparticle drug carriers including liposomeformulations as a typical example and polypeptides of proteinpreparations and the like have poor retention in blood after intravenousadministration and are readily captured by the reticuloendothelialsystem (henceforth abbreviated as “RES”) of liver, spleen and the like.The presence of RES is a serious obstacle when preparations are utilizedsuch as targeting type preparations which can deriver medicaments toorgans other than RES, or microparticle drug carriers as sustainedrelease type preparations which can provide long term blood retention ofmedicaments and achieve control release of the medicaments.

Researches have been conducted so far to impart microcirculatability tothe aforementioned preparations. For example, from a standpoint thatphysicochemical properties of lipid bimolecular membranes of liposomesare relatively easily controllable, some methods have been proposed suchas a method of increasing blood level of liposomes by using a smallersize of liposomes (Biochimica et Biophysica Acta, vol. 761, p. 142,1983), a method of using lecithin having a high phase transitiontemperature (Biochemical Pharmacology, vol. 32, p. 3381, 1983), a methodof using sphingomyelin instead of lecithin (Biochemical Pharmacology,vol. 32 volumes, p. 3381, 1983), and a method of adding cholesterol as amembrane component of liposomes (Biochimica et Biophysica Acta, vol.761, p. 142, 1983). However, among the aforementioned methods, no methodis known to successfully provide microparticle drug carrier which hasexcellent retention in blood and is hardly taken up by RES.

As other solutions, researches have been conducted for impartingmicrocirculatability and avoiding RES by membrane surfaces modificationof liposomes with glycolipids, glycoproteins, amino acid lipids,polyethylene glycol lipids or the like. For example, those reported assubstances for such modification include glycophorin (PharmaceuticalSociety of Japan, 106th Annual Convention, Lecture Abstracts, p. 336,1986), ganglioside GM1 (FEBS letter, vol. 223, p. 42, 1987),phosphatidylinositol (FEBS Letter, vol. 223, p. 42, 1987), glycophorinand ganglioside GM3 (Japanese Patent Unexamined Publication (Kokai) No.63-221837), polyethylene glycol derivative (FEBS Letter, vol. 268, p.235, 1990), glucuronic acid derivative (Chemical and PharmaceuticalBulletin, vol. 38, p. 1633, 1990), glutamic acid derivative (Biochimicaet Biophysica Acta, vol. 1108, p. 257, 1992), polyglycerin phospholipidderivative (Japanese Patent Unexamined Publication No. 6-228012) and thelike.

As for the modification of polypeptide, introduction of twowater-soluble polymer molecules by using triazine and other methods havebeen reported in order to reduce binding sites of a polypeptide andthereby increase an amount of remaining active groups such as lysineresidues in the polypeptide. Also as for liposome preparations, a methodis reported in which two water-soluble polymer molecules are introducedinto triazine to increase molecular weight of the water-solublepolymers, and liposome surfaces are modified by using the polymers. Thenumber of the water-soluble polymers in said modification is limited upto 2. It is considered that, in this attempt, an effect of impartingmicrocirculatability to liposome surfaces is lower than liposomes withthe hydrophilic groups. Furthermore, although phospholipid derivativescontaining a polyalkylene oxide group have been used also assurfactants, no derivative has been known which is highly safe forliving bodies and stably usable under a high salt concentrationcondition.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a novel phospholipidderivative containing a polyalkylene oxide. More specifically, theobject of the present invention is to provide a phospholipid derivativewhich is highly safe for living bodies, and is suitably used forsolubilization and dispersion of physiologically active substances andthe like, or in the fields of drug delivery systems such as liposomesand cosmetics. The inventors of the present invention conducted variousstudies to achieve the foregoing object. As a result, the inventorssuccessfully provided novel phospholipid derivatives represented by thefollowing formula and a method for producing the same.

The present invention thus provides a phospholipid derivativerepresented by the following general formula (1):

wherein Z represents a residue of a compound having 3 to 10 hydroxylgroups; AO represents an oxyalkylene group having from 2 to 4 carbonatoms; R¹CO and R²CO independently represent an acyl group having 8 to22 carbon atoms; X represents hydrogen atom, an alkali metal atom,ammonium or an organic ammonium; “a” represents an integer of 0 to 4;“b” represents 0 or 1; Q represents hydrogen atom or methyl group; mrepresents an average number of moles of the oxyalkylene group added;and m, k1, k2, and k3 are numbers satisfying the following conditions:3≦m≦200, 9≦m×(k1+k2+k3)≦1000, 1≦k1≦2, 0≦k2≦9 and 0≦k3≦9, and3≦k1+k2+k≦10.

According to preferred embodiments of the above present invention,provided are the aforementioned phospholipid derivative, wherein thecondition 4≦k1+k2+k3≦8 is satisfied; the aforementioned phospholipidderivative, wherein R¹CO and R²CO independently represent an acyl grouphaving from 12 to 20 carbon atoms; the aforementioned phospholipidderivative, wherein k2 is 0; the aforementioned phospholipid derivative,wherein “a” and “b” represent 0; and the aforementioned phospholipidderivative, wherein the following conditions k3<1 and k2>k3 aresatisfied.

From another aspect of the present invention, provided are a surfactantcomprising a phospholipid derivative represented by the aforementionedgeneral formula (1); a solubilizer comprising a phospholipid derivativerepresented by the aforementioned general formula (1); a dispersingagent, preferably a dispersing agent for cosmetics, comprising aphospholipid derivative represented by the aforementioned generalformula (1); and a lipid membrane structure, preferably liposome,comprising a phospholipid derivative represented by the aforementionedgeneral formula (1).

From a further aspect of the present invention, provided is a method forproducing a phospholipid derivative represented by the aforementionedgeneral formula (1), which comprises the step of reacting a phospholipidcompound represented by the following general formula (2):

wherein R¹CO and R²CO independently represent an acyl group having from8 to 22 carbon atoms; X represents hydrogen atom, an alkali metal atom,ammonium or an organic ammonium; “a” represents an integer of from 0 to4; and Y represents hydrogen atom or N-hydroxysuccinimide, and apolyalkylene oxide compound represented by the following general formula(3):Z-[—O(AO)_(m)—H]_(k)  (3)wherein Z represents a residue of a compound having from 3 to 10hydroxyl groups; AO represents one or two or more kinds of oxyalkylenegroups having from 2 to 4 carbon atoms, and when AO represents two ormore kinds of oxyalkylene groups, they may bond to form a blockcopolymer or random copolymer; “m” represents an average number of molesof the oxyalkylene group added; and m and k are numbers satisfying thefollowing conditions: 3≦m≦200, 9≦m×k≦1000, and 3≦k≦10, in an organicsolvent in the presence of a basic catalyst (this step is also referredto as “Step A”). This method can be preferably performed at atemperature in the range of from 20 to 90° C., and is preferablyperformed in the presence of a dehydrocondensing agent.

Further, also provided is a method for producing a phospholipidderivative represented by the aforementioned general formula (1), whichcomprises the step of reacting a polyalkylene oxide derivativerepresented by the following general formula (4):

wherein Z represents a residue of a compound having from 3 to 10hydroxyl groups; “a” represents an integer of from 0 to 4; “b”represents 0 or 1; “m” represents an average number of moles of theoxyalkylene group added; Y represents hydrogen atom orN-hydroxysuccinimide; and k4 and k5 are numbers satisfying the followingconditions: 1≦k4≦10, 0≦k5≦9, and 3≦k4+k5≦10, and a phospholipidderivative represented by the following general formula (5):

wherein R¹CO and R²CO have the same meanings as those defined in theaforementioned formula (1), in organic solvents in the presence of abasic catalyst. This method can be preferably performed at a temperaturein the range of from 20 to 90° C.

BEST MODE FOR CARRYING OUT THE INVENTION

In the phospholipid derivatives of the present invention represented bythe formula (1), Z is a residue of a compound having from 3 to 10hydroxyl groups. A type of the compound having from 3 to 10 hydroxylgroups is not particularly limited. Examples include, for example,glycerin, polyglycerin compounds such as diglycerin, pentaerythritol,triglycerin, tetraglycerin, pentaglycerin, hexaglycerin, heptaglycerin,and octaglycerin. In the specification, the residue of a compound havingfrom 3 to 10 hydroxyl groups means a residual portion of the compoundobtained by eliminating hydroxyl groups of a total number (k1+k2+k3)wherein each of k1, k2 and k3 represents arms.

The value of k1+k2+k3 corresponds to the number of arms on Z, and thenumber is an integer in the range of from 3 to 10, preferably from 3 to8, more preferably from 4 to 8 (the numerical ranges indicated with“from—to” in the specification are ranges including numerical values ofupper and lower limits). When the number of arms is less than 3, thedesired effect of the compounds may sometimes not be obtained. When thenumber of arms is more than 10, viscosity of multi-arm raw materials,including polyglycerine and the like as typical examples, becomes high,which causes difficulty in handling the materials. In addition, the rawmaterials may sometimes be hardly obtainable.

The symbol k1 represents the number of a partial structure, containing aresidue of a phospholipid compound represented by the formula (2), thatbonds to a residue represented by Z, and the number is 1 or 2. When thenumber of the partial structure containing the aforementionedphospholipid compound is 0, the compound cannot stably bind to lipidbilayer membranes such as those of liposomes because no hydrophobic bondportion exists. Therefore, modification of liposome membranes with sucha compound may become difficult. Further, when the number of the partialstructure containing the aforementioned phospholipid compound is largerthan 2, many phospholipid residues are contained in a single molecule,and hydrophobic bonding power to liposome membranes may become stronger.Therefore, the degree of freedom of the polyoxyethylene chain may becomesmall, and thus the desired effect of the compounds of the presentinvention may sometimes not be obtained.

R¹CO and R²CO independently represent an acyl group having from 8 to 22carbon atoms, preferably from 12 to 20 carbon atoms, more preferablyfrom 14 to 18 carbon atoms. A type of the acyl group is not particularlylimited, and any of an aliphatic acyl group or an aromatic acyl groupmay be used. In general, an acyl group derived from an aliphatic acidmay preferably be used. Specific examples of R¹CO and R²CO include, forexample, acyl groups derived from saturated or unsaturated linear orbranched aliphatic acids such as caprylic acid, capric acid, lauricacid, myristic acid, palmitic acid, palmitoleic acid, stearic acid,isostearic acid, oleic acid, linolic acid, arachic acid, behenic acid,erucic acid and lignoceric acid. R¹CO and R²CO may be the same ordifferent. When the carbon number of R¹CO and R²CO exceeds 22,dispersibility of the compounds in an aqueous phase may become poor, andreactivity may sometimes be lowered. When the carbon number is less than8, the crystallinity may become poor in a purification step, and a finalpurity of a target substance may become low.

Symbol k2 represents the number of the partial structure, of which endis represented by —COOX, that bonds to a residue represented by Z, andthe number is chosen from the range of from 0 to 9. When the number is0, it is meant that no partial structure of which end is represented by—COOX substantially exists in the compounds of the present invention. Xrepresents hydrogen atom, an alkali metal atom, ammonium or an organicammonium, preferably hydrogen atom or an alkali metal atom. Specificexamples of the alkali metal atom include sodium, potassium and thelike, and specific examples of the organic ammonium includetriethylammonium and the like.

Symbol k3 represents the number of the partial structure, of which endis hydroxyl group or methyl group, that bonds to a residue representedby Z, and the number is chosen from the range of from 0 to 9. Q ishydrogen atom or methyl group. When Q is an alkyl group other thanmethyl group, hydrophilicity of the compounds of the present inventionmay sometimes be lost.

Symbol b is an integer of 0 or 1, and when “b” is 1, “a” is preferablyan integer of from 1 to 4, more preferably 2 or 3. When “b” is 0, “a” ispreferably 1 or 0, more preferably 0.

The oxyalkylene group represented by AO is an oxyalkylene group havingfrom 2 to 4 carbon atoms, preferably 2 or 3 carbon atoms, and examplesinclude, for example, oxyethylene group, oxypropylene group,oxytrimethylene group, oxybutylene group and the like. Among them,oxyethylene group and oxypropylene group are preferred, and oxyethylenegroup is particularly preferred. The oxyalkylene groups that constitutethe polyoxyalkylene group represented by -(AO)_(m)— may consist of asingle kind of oxyalkylene groups. Alternatively, they may consist of acombination of two or more kinds of oxyalkylene groups. When two or morekinds of oxyalkylene groups are combined, a manner of combination is notparticularly limited, and the polyoxyalkylene group may be a block orrandom copolymer. When a ratio of oxyethylene groups based on totaloxyalkylene groups is low, water solubility may sometimes be lowered.Therefore, the ratio of oxyethylene groups based on the totaloxyalkylene groups is preferably from 50 to 100 mole %.

Symbol m represents an average number of moles of the oxyalkylene groupadded, and the number is from 8 to 200, preferably from 7 to 80. When mis smaller than 3, a desired effect of the phospholipid derivatives ofthe present invention, as used in a drug delivery system, may bereduced. When the number is larger than 200, reactivity between aphospholipid compound represented by the formula (2) and a polyalkyleneoxide compound represented by the formula (3) may be reduced in thepreparation of the phospholipid derivatives of the present invention,and viscosity of a polyalkylene oxide compound represented by theformula (3) may be increased, which may sometimes result in degradationof workability. In addition, symbol m means the number of oxyalkylenegroups contained in polyoxyalkylene group existing in each of [k1+k2+k3]of arms contained in compounds of the present invention. Formulam×[k1+k2+k3] means the number of oxyalkylene groups contained in thecompounds of the present invention as a whole, and the number is from 9to 1000, preferably from 20 to 700, more preferably from 30 to 350.

The method for producing the compounds of the present inventionrepresented by the formula (1) is not particularly limited. Thephospholipid derivatives wherein k2 is 0 can be produced, for example,by Step A with a high purity. In the phospholipid compounds representedby the formula (2) used in Step (A), R¹CO, R²CO and “a” are the same asthose explained in the formula (1), and Y is hydrogen atom orN-hydroxysuccinimide.

The phospholipid compounds represented by the formula (2) can beproduced by a known method. For example, the compounds can be easilyproduced by reacting a phospholipid compound with a dicarboxylic acidanhydride as described later. The phospholipid to be used may be anatural phospholipid or a synthetic phospholipid, and examples include,for example, synthetic and natural phosphatidylethanolamines such assoybean phosphatidyldiethanolamine, hydrogenated soybeanphosphatidyldiethanolamine, egg yolk phosphatidyldiethanolamine andhydrogenated egg yolk phosphatidyldiethanolamine, and the like.

In the polyalkylene oxide compounds represented by the formula (3) usedin Step (A), Z, AO and m are the same as those explained as for theformula (1). Symbol k corresponds to the sum of k1, k2 and k3, eachexplained as for the formula (1). The reaction of a polyalkylene oxidecompound represented by the formula (3) and a phospholipid compoundrepresented by the formula (2) can be performed in organic solvents inthe presence of a basic catalyst, and the reaction can be generallycarried out by using a dehydrocondensing agent.

A type of the basic catalyst is not particularly limited. Examplesinclude, for example, as nitrogen-containing substances, triethylamine,pyridine, dimethylaminopyridine, ammonium acetate and the like, asorganic salts, sodium phosphate, sodium carbonate, sodiumhydrogencarbonate, sodium borate, sodium acetate and the like. Theamount of the basic catalyst is, for example, from 1.5 to 10 moles,preferably from 2 to 5 moles, per mole of the polyalkylene oxidecompound represented by the formula (3). As the organic solvent, thosenot having a reactive functional group such as hydroxyl group can beused without any particular limitation. Examples include, for example,ethyl acetate, dichloromethane, chloroform, benzene, toluene and thelike. Among them, chloroform and toluene are preferred. Organic solventshaving hydroxyl group, such as ethanol, may sometimes react with thecarboxyl group at the end of the phospholipid compound represented bythe formula (2).

When a dehydrocondensing agent is used, those achieving dehydrationcondensation of the hydroxyl group of the polyalkylene oxide compoundrepresented by the formula (3) and a carboxylic acid group of thephospholipid represented by the formula (2) can be used without anyparticular limitation. Examples of such dehydrocondensing agents includecarbodiimide derivatives such as dicyclohexylcarbodiimide, anddicyclohexylcarbodiimide is particularly preferred. The amount of thedehydrocondensing agent used is, for example, desirably from 1.05 to 5moles, preferably from 1.5 to 2.5 moles, per mole of the polyalkyleneoxide compound represented by the formula (3). An yield may sometimes beincreased by adding N-hydroxysuccinimide to the reaction system in anamount of from 0.1 to 2 moles per mole of the polyalkylene oxidecompound represented by the formula (3).

The reaction temperature of Step (A) is usually in the range of from 20to 90° C., preferably from 40 to 80° C. The reaction time is desirably 1hour or more, preferably from 2 to 8 hours. At a temperature lower than20° C., a reactivity may sometimes become lowered, and at a temperaturehigher than 90° C., the acyl group of the phospholipid compoundrepresented by the formula (2) used for the reaction may sometimes behydrolyzed.

The compounds of the present invention represented by the formula (1)can also be produced by reacting an activated ester derivative of thephospholipid compound represented by the formula (2) and a polyalkyleneoxide compound represented by the formula (3). The aforementionedactivated ester derivative can be obtained by reacting, for example, aphospholipid compound represented by the formula (2) and an activatingagent in the presence of a dehydrocondensing agent. A type of theaforementioned activating agent is not particularly limited, andexamples include, for example, N-hydroxysuccinimide, N,N′-disuccinimidecarbonate, 1-hydroxybenzotriazole,N-hydroxy-5-norbornene-2,3-dicarboxyimide, N-hydroxyphthalimide,4-hydroxyphenyldimethylsulfonium methyl sulfate, isobutyl chloroformateand the like. Among these, N-hydroxysuccinimide is preferred.

The reaction of the phospholipid compound represented by the formula (2)and the activating agent can be performed, for example, at a reactiontemperature of from 15 to 80° C., preferably from 25 to 55° C. in thepresence of a dehydrocondensing agent in a solvent that does not reactwith carboxylic acids, such as chloroform and toluene, in the samemanner as the reaction with a dicarboxylic acid anhydride. The reactioncan be performed by, for example, dispersing the activating agent in asolution of the polyalkylene oxide compound and stirring the dispersion.For example, when N-hydroxysuccinimide is used as the activating agent,the carboxyl group of the phospholipid compound represented by theformula (2) and the hydroxyl group of N-hydroxysuccinimide react to givean activated ester derivative consisting of the phospholipid compoundrepresented by the formula (2) to which N-hydroxysuccinimide bonds atthe end of the carboxyl group side of the phospholipid compoundrepresented by the formula (2).

The phospholipid derivatives of the formula (1) wherein k2 is not 0,i.e., the compounds which have the partial structure of branchingoxyalkylene group having carboxyl group at the end, can be produced witha high purity by reacting a polyalkylene oxide derivative represented bythe aforementioned general formula (4) and a phospholipid derivativerepresented by the aforementioned general formula (5) in an organicsolvent in the presence of a basic catalyst.

As the organic solvent used for the reaction, those not having areactive functional group such as hydroxyl group can be used without anyparticular limitation. Examples include, for example, ethyl acetate,dichloromethane, chloroform, benzene, toluene and the like. Among them,chloroform and toluene are preferred. Organic solvents having hydroxylgroup such as ethanol may sometimes react with the carboxyl group at theend of the polyalkylene oxide compound represented by the formula (4).

A type of the basic catalyst used for the reaction is not particularlylimited. Examples include, for example, as nitrogen-containingsubstances, triethylamine, ammonium acetate and the like, as organicsalts, sodium phosphate, sodium carbonate, sodium hydrogencarbonate,sodium borate, sodium acetate and the like. The amount of the basiccatalyst is, for example, from 1.5 to 10 moles, preferably from 2 to 7moles, per mole of the polyalkylene oxide compound represented by theformula (4). The reaction temperature is usually from 20 to 90° C.,preferably from 40 to 80° C. The reaction time is 1 hour or more,preferably from 2 to 8 hours. At a temperature lower than 20° C., areactivity may sometimes become lowered, and at a temperature higherthan 90° C., the acyl group of the phospholipid compound represented bythe formula (5) used for the reaction may sometimes be hydrolyzed. Thecompounds of the present invention may be obtained as a single kind ofcompound, or alternatively, may be obtained as a mixture of compoundshaving different numbers for each of k1, k2 and k3 depending on thesynthetic procedures. Such a mixture is also fall within the scope ofthe present invention.

By using the compounds of the present invention represented by theaforementioned general formula (1) as surfactants, a solubilizedsolution, an emulsion, and a dispersion can be obtained. When thesurfactant of the present invention is used as an emulsifier, asolubilizer, or a dispersing agent, the emulsifier, solubilizer, ordispersing agent may solely contain the surfactant of the presentinvention, or alternatively, the emulsifier, solubilizer or dispersingagent may contain other known ingredients which are used foremulsification, solubilization, or dispersion. A form of the solubilizedsolution or dispersion is not limited. Examples include solutions inwhich a fat-soluble substance or the like is dissolved in a medium suchas water and buffers, dispersions in which a fat-soluble substance orthe like is dispersed in a medium such as water and buffers, and thelike.

A form of the emulsion or solubilized solution is not limited. Examplesinclude micellar solutions formed with the surfactant of the presentinvention, i.e., micellar solutions in which a fat-soluble substance iscontained in micelles, emulsion solutions in which dispersed particles,which are formed with the surfactant of the present invention and afat-soluble substance or the like, exist in a dispersion medium such aswater and buffers as colloidal particles or larger particles. Examplesof the micellar solutions include, in particular, polymer micellarsolutions in which dispersed particles have a diameter of 10 to 300 nm.The emulsion solutions may an O/W type emulsion in which a fat-solublesubstance is added to an oil phase, or a W/O/W type emulsion in which afat-soluble substance is added to an aqueous phase. The fat-solublesubstance that can be solubilized or emulsified is not particularlylimited. Examples include, for example, higher alcohols, ester oils,triglycerin, tocopherol, higher fatty acids, phospholipids, hardlysoluble medicaments such as adriamycin, cisplatin, paclitaxel, andamphotericin B. An application as a dispersing agent in the cosmeticfield is also not particularly limited. When a water-soluble substancesuch as ascorbic acid should be retained in an internal aqueous phase ofa lipid membrane structure, or when a fat-soluble substance such astocopherol should be retained in a lipid bilayer membrane, the objectivesubstance can be more stably dispersed in an aqueous solution by usingthe compounds of the present invention as an agent for forming a lipidmembrane structure. When the subject compounds are used as surfactantsor dispersing agents, an amount of 0.1 to 20% by weight, preferably 0.5to 7% by weight, more preferably 0.5 to 5% by weight, may be used basedon the total weight of the substances to be solubilized, dispersed,emulsified or the like.

The compounds of the aforementioned general formula (1) wherein k2 is 0can be effectively used, in particular, as nonionic surfactants under ahigh salt concentration. Polyalkylene oxide-modified phospholipids andthe like generally have hydrophilicity deriving from oxyalkylene groupsand hydrophobicity deriving from acyl groups, and accordingly, they canbe used as surfactants. However, the surfactants having oxyalkylenegroups, including polyalkylene oxide-modified phospholipids as typicalexamples, generally have a problem in that they generate turbidity whenused under a condition of a high salt concentration. Moreover, althougha use of nonionic surfactants consisting of glycidol derivatives under ahigh salt concentration condition was reported, such use causes problemsof skin irritation due to remaining glycidyl compounds and the like, andaccordingly, they have a problem in that they are not suitable for usein the cosmetic field. The compounds of the aforementioned generalformula (1) wherein Q is hydrogen atom have a characteristic propertythat they can maintain a high solubilizing ability even under a highsalt concentration condition, and can be used as surfactants havingsuperior salt tolerance. They can also be used as surfactants havingsuperior compatibility with skins in the cosmetic field.

The compounds of the aforementioned general formula (1) wherein k3<1 andk2>k3, i.e., the compounds having carboxyl group at the end of the armsof polyoxyalkylene chain, can be used, for example, as dispersing agentsas pH-sensitive phospholipids. When a cationic substance (e.g., cationicphysiologically active substances and the like), basic substance or thelike is dispersed in water, stable dispersion in water can be obtainedby coating the surfaces of microparticles containing the cationicsubstance, basic substance or the like with the aforementionedcompounds. The compounds of the present invention have a polyanionicgroup, and accordingly, they can provide stable dispersion on the basisof ionic bonds. When Q is hydrogen atom, and both of k2 and k3 are 1 ormore, carboxyl groups and the like coexist with hydroxyl groups, andsuch compounds may be crosslinked by intermolecular bonds and gelledduring a manufacturing process. For this reason, when the compounds areused as anionic dispersing agents, k3 is preferably smaller than 1.

The compounds of the present invention represented by the aforementionedgeneral formula (1) can be used as phospholipids constituting lipidmembrane structures such as liposomes, emulsions, and micelles. By usingthe compounds of the present invention, circulation time in blood oflipid membrane structures, preferably liposomes, can be increased. Thiseffect can be attained by adding a small amount of the compounds of thepresent invention to the lipid membrane structures. Although it is notintended to be bound by any specific theory, by using the compounds ofthe present invention having three or more arms as a phospholipidconstituting a lipid membrane structure, aggregation of microparticlesin an aqueous solution is prevented, and a stable dispersed state isattained, because the polyoxyethylene chains have three-dimensionalbulkiness in the membranes of the lipid membrane structures.

An amount of the compounds of the present invention mixed in lipidmembrane structures may be an amount sufficient for effective expressionof efficacy of a medicament in a living body, and the amount is notparticularly limited. For example, the amount can be suitably chosendepending on a type of a medicament to be contained in lipid membranestructures, a purpose of use such as therapeutic use and prophylacticuse, a form of lipid membrane structures and the like. A type of amedicament contained in lipid membrane structures provided by thepresent invention is not particularly limited. For example, compoundsused as antitumor agents are preferred. Examples of such compoundsinclude, for example, irinotecan hydrochloride, nogitecan hydrochloride,exatecan, RFS-2000, Lurtotecan, camptothecin derivatives such asBNP-1350, Bay-383441, PNU-166148, IDEC-132, BN-80915, DB38, DB-81,DB-90, DB-91, CKD-620, T-0128, ST-1480, ST-1481, DRF-1042 and DE-310,docetaxel hydrate, paclitaxel, taxane derivatives such as IND-5109,BMS-184476, BMS-188797, T-3782, TAX-1011, SB-RA-31012, SBT-1514 andDJ-927, ifosphamide, nimustine hydrochloride, carboquone,cyclophosphamide, dacarbazine, thiotepa, busulfan, melphalan,ranimustine, estramustine phosphate sodium, 6-mercaptopurine riboside,enocitabine, gemcitabine hydrochloride, carmofur, cytarabine, cytarabineocphossfate, tegafur, doxifluridine, hydroxycarbamide, fluorouracil,methotrexate, mercaptopurine, fludarabine phosphate, actinomycin D,aclarubicin hydrochloride, idarubicin hydrochloride, epirubicinhydrochloride, daunorubicin hydrochloride, doxorubicin hydrochloride,pirarubicin hydrochloride, bleomycin hydrochloride, zinostatinstimalamer, neocarzinostatin, mitomycin C, bleomycin sulfate, peplomycinsulfate, etoposide, vinorelbine ditartrate, vincristine sulfate,vindesine sulfate, vinblastine sulfate, amrubicin hydrochloride,gefitinib, exemestane, capecitabine, TNP-470, TAK-165, KW-2401, KW-2170,KW-2871, KT-5555, KT-8391, TZT-1027, S-3304, CS-682, YM-511, YM-598,TAT-59, TAS-101, TAS-102, TA-106, FK-228, FK-317, E7070, E7389, KRN-700,KRN-5500, J-107088, HMN-214, SM-11355, ZD-0473 and the like.

Further, a gene or the like may be encapsulated in the lipid membranestructures of the present invention. The gene may be any ofoligonucleotide, DNA, and RNA, and examples include, in particular,genes for in vitro gene transfer such as transformation, genes actingupon in vivo expression, for example, genes for gene therapies and genesused for breeding of industrial animals such as laboratory animals andfarm animals. Examples of the genes for gene therapies include antisenseoligonucleotides, antisense DNAs, antisense RNA., genes coding forphysiologically active substances such as enzymes and cytokines, and thelike.

The aforementioned lipid membrane structures may be further added withphospholipids, sterols such as cholesterol and cholestanol, otheraliphatic acids having a saturated or unsaturated acyl group of 8 to 22carbon atoms, and antioxidants such as α-tocopherol in addition to thecompound of the present invention. Examples of the phospholipids includephosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,phosphatidylinositol, phosphatidylglycerol, cardiolipin, sphingomyelin,ceramide phosphorylethanolamine, ceramide phosphorylglycerol, ceramidephosphorylglycerol phosphate,1,2-dimyristoyl-1,2-deoxyphosphatidylcholine, plasmalogen, phosphatidicacid and the like, and these substances can be used alone or incombination of two or more kinds. The aliphatic acid residues of thesephospholipids are not particularly limited. Examples include saturatedor unsaturated aliphatic acid residues having 12 to 20 carbon atoms, andspecific examples include, for example, acyl groups derived fromaliphatic acids such as lauric acid, myristic acid, palmitic acid,stearic acid, oleic acid, and linolic acid. Moreover, phospholipidsderived from natural products such as egg yolk lecithin and soybeanlecithin can also be used.

Forms of lipid membrane structures containing the compounds of thepresent invention and methods for preparation thereof are notparticularly limited. Examples of available forms thereof include, forexample, forms of dried lipid mixtures, forms of dispersions in aqueoussolvents, dried or solidified forms of these and the like. When apreparation in a form of dried lipid mixture is desired, the form can beprepared by, for example, once dissolving lipid components to be used inan organic solvent such as chloroform and then subjecting the solutionto solidification under reduced pressure using an evaporator or tospray-drying by using a spray dryer. Examples of the lipid membranestructures dispersed in an aqueous solvent include single membraneliposomes, multilayer liposomes, O/W type emulsions, W/O/W typeemulsions, spherical micelles, fibrous micelles, layered structures ofirregular shapes and the like. Among them, liposomes are preferred. Asize of the lipid membrane structures in a dispersed state is notparticularly limited. For example, liposomes and emulsions may have aparticle diameter of 50 nm to 5 μm, and spherical micelles may have aparticle diameter of 5 nm to 100 nm. As for fibrous micelles and layeredstructures of irregular shapes, they are considered to be constructedwith layers which has a thickness of 5 to 10 nm per layer.

Components of the aqueous solvent (medium) is also not particularlylimited, and the medium may be a buffer such as phosphate buffer,citrate buffer and phosphate buffered physiological saline,physiological saline, cell culture medium or the like. When thecompounds of the present invention are used in an aqueous solvent, thelipid membrane structures can be stably dispersed. Besides water, anaqueous solution of saccharide such as glucose, lactose and sucrose, anaqueous solution of polyhydric alcohol such as glycerin and propyleneglycol or the like may be added. In order to stably store the lipidmembrane structures dispersed in such an aqueous solvent for a longperiod of time, it is desirable to eliminate electrolytes in the aqueoussolvent as low as possible from a viewpoint of physical stability, forexample, prevention of aggregation. Moreover, from a viewpoint ofchemical stability of lipids, it is desirable to adjust pH of theaqueous solvent to be within the range of weakly acidic to approximatelyneutral pH (pH 3.0 to 8.0), or to remove dissolved oxygen by nitrogenbabbling. Further, when the lipid membrane structures are stored afterlyophilization or spray drying, for example, use of each of a saccharideaqueous solution and a polyhydric alcohol aqueous solution inlyophilization of a saccharide aqueous solution may achieve effectivepreservation. A concentration of these aqueous solvents is notparticularly limited. For example, the concentration of the saccharideaqueous solution may preferably be 2 to 20% (W/V), more preferably 5 to10% (W/V). Also for example, the concentration of the polyhydric alcoholaqueous solution may preferably be 1 to 5% (W/V), more preferably 2 to2.5% (W/V). In the buffers, a concentration of buffering agent maypreferably be 5 to 50 mM, more preferably 10 to 20 mM. A concentrationof the lipid membrane structures in the aqueous solvent is notparticularly limited. The concentration of the total lipids in the lipidmembrane structures may preferably be 0.1 to 500 mM, more preferably 1to 100 mM.

The lipid membrane structures in a form of a dispersion in an aqueoussolvent can be prepared by adding the aforementioned dried lipid mixtureto the aqueous solvent and emulsifying the mixture by using anemulsifier such as homogenizer, ultrasonic emulsifier, high-pressureinjection emulsifier or the like. They can also be produced by a methodwell known as a method for producing liposomes, for example, the reversephase evaporation method or the like, and the methods are notparticularly limited. When a control of the size of the lipid membranestructures is desired, extrusion (extrusion filtration) can be performedunder a high pressure by using a membrane filter having uniform poresizes.

Examples of a method for further drying the aforementioned lipidmembrane structures dispersed in an aqueous solvent include ordinarylyophilization and spray drying. As the aqueous solvent used for thesemethods, an aqueous solution of saccharide, preferably aqueous solutionof sucrose or aqueous solution and lactose, may be used as describedabove. When lipid membrane structures dispersed in an aqueous solventare once produced and further dried, long-term storage of the lipidmembrane structures becomes possible. In addition, when an aqueoussolution of a medicament is added to such dried lipid membranestructures, the lipid mixture is efficiently hydrated, and thus anadvantage is provided that a medicament can be efficiently retained inthe lipid membrane structures. By adding a medicament to the lipidmembrane structures, a pharmaceutical composition can be produced, andthe resulting lipid membrane structures can be used as a pharmaceuticalcomposition for therapeutic treatment and/or prophylactic treatment of adisease. When the medicament is a gene, the composition can be used fora kit for gene transfer.

As for a form of the pharmaceutical composition, the form may be thelipid membrane structures retaining a medicament, as well as a mixtureof a medicament and the lipid membrane structures. The term “retain”used herein means that a medicament exists inside the membranes of thelipid membrane structures, on the membrane surfaces, in the membranes,in the lipid layers, and/or on the lipid layer surfaces. An availableform of the pharmaceutical composition and a method for preparationthereof are not particularly limited in the same manner as the lipidmembrane structures. As for the available form, examples include a formof a dried mixture, a form of a dispersion in an aqueous solvent, andforms obtained by further drying or freezing said forms.

A dried mixture of lipids and a medicament can be produced by, forexample, once dissolving lipid components and a medicament to be used inan organic solvent such as chloroform and then subjecting the resultingsolution to solidification under reduced pressure by using an evaporatoror spray drying by using a spray dryer. Examples of a form in which amixture of lipid membrane structures and a medicament are dispersed inan aqueous solvent include, but not particularly limited thereto,multi-layer liposomes, single membrane liposomes, O/W type emulsions,W/O/W type emulsions, spherical micelles, fibrous micelles, layeredstructures of irregular shapes and the like. A size of particles(particle diameter) as the mixture, a composition of the aqueous solventand the like are not particularly limited. For example, liposomes mayhave a size of 50 nm to 2 μm, spherical micelles may have a size of 5 to100 nm, and emulsions may have a particle diameter of 50 nm to 5 μm. Aconcentration of the mixture in the aqueous solvent is also notparticularly limited. Several methods are known as methods for producinga mixture of lipid membrane structures and a medicament in the form ofdispersion in an aqueous solvent. It is necessary to appropriately chosea suitable method depending on an available form of the mixture of lipidmembrane structures and a medicament.

Production Method 1

Production Method 1 is a method of adding an aqueous solvent to theaforementioned dried mixture of lipids and a medicament and emulsifyingthe mixture by using an emulsifier such as homogenizer, ultrasonicemulsifier, high-pressure injection emulsifier, or the like. If it isdesired to control the size (particle diameter), extrusion (extrusionfiltration) can be further performed under a high pressure by using amembrane filter having uniform pore sizes. In this method, in order toprepare a dried mixture of lipids and a medicament first, it isnecessary to dissolve the medicament in an organic solvent, and themethod has an advantage that it can make the best utilization ofinteractions between the medicament and lipid membrane structures. Evenwhen the lipid membrane structures have a multi-layer structure, amedicament can enter into the inside of the multiple layers, and thususe of this method generally provides a higher retention ratio of themedicament in the lipid membrane structures.

Production Method 2

Production Method 2 is a method of adding an aqueous solvent containinga medicament to dried lipid components obtained by dissolving the lipidcomponents in an organic solvent and evaporating the organic solvent,and emulsifying the mixture. If it is desired to control the size(particle diameter), extrusion (extrusion filtration) can be furtherperformed under a high pressure by using a membrane filter havinguniform pore sizes. This method can be used for a medicament that ishardly dissolved in an organic solvent, but can be dissolved in anaqueous solvent. When the lipid membrane structures are liposomes, theyhave an advantage that they can retain a medicament also in the part ofinternal aqueous phase.

Production Method 3

Production Method 3 is a method of further adding an aqueous solventcontaining a medicament to lipid membrane structures such as liposomes,emulsions, micelles or layered structures already dispersed in anaqueous solvent. This method is limitedly applied to a water-solublemedicament. The addition of a medicament to already prepared lipidmembrane structures is performed from the outside. Therefore, if themedicament is a polymer, the medicament cannot enter into the inside ofthe lipid membrane structures, and the medicament may be present in aform that it binds to the surfaces of lipid membrane structures. Whenliposomes are used as the lipid membrane structures, use of ProductionMethod 3 may result in formation of a sandwich-like structure in whichthe medicament is sandwiched between liposome particles (generallycalled as a complex). An aqueous dispersion of lipid membrane structuresalone is prepared beforehand in this production method. Therefore,decomposition of a medicament during the preparation need not be takeninto consideration, and a control of the size (particle diameter) isalso readily operated, which enables relatively easier preparationcompared with Production Methods 1 and 2.

Production Method 4

Production Method 4 is a method of further adding an aqueous solventcontaining a medicament to a dried product obtained by once producinglipid membrane structures dispersed in an aqueous solvent and thendrying the same. In this method, a medicament is limited to awater-soluble medicament in the same manner as Production Method 3. Asignificant difference from Production Method 3 is a mode of presence ofthe lipid membrane structures and a medicament. That is, in ProductionMethod 4, lipid membrane structures dispersed in an aqueous solvent areonce produced and further dried to obtain a dried product, and at thisstage, the lipid membrane structures are present in a state of a solidas fragments of lipid membranes. In order to allow the fragments oflipid membranes to be present in a solid state, it is preferable to usean aqueous solution of a saccharide, preferably an aqueous solution ofsucrose or aqueous solution of lactose, as the aqueous solvent asdescribed above. In this method, when the aqueous solvent containing amedicament is added, hydration of the fragments of the lipid membranespresent in a state of a solid quickly starts with the invasion of water,and thus the lipid membrane structures can be reconstructed. At thistime, a structure of a form in which a medicament is retained in theinside of the lipid membrane structures can be produced.

In Production Method 3, when a medicament is a polymer, the medicamentcannot enter into the inside of the lipid membrane structures, and ispresent in a mode that it binds to the surfaces of the lipid membranestructures. Production Method 4 significantly differs in this point. InProduction Method 4, an aqueous dispersion of lipid membrane structuresalone is prepared beforehand, and therefore, decomposition of themedicament during the emulsification need not be taken intoconsideration, and a control of the size (particle diameter) is alsoeasy attainable. For this reason, said method enables relatively easierpreparation compared with Production Methods 1 and 2. Besides the abovementioned advantages, this method also has advantages that storagestability for a pharmaceutical preparation is easily secure, because themethod uses lyophilization or spray drying; when the dried preparationis rehydrated with an aqueous solution of a medicament, original size(particle diameter) can be reproduced; when a polymer medicament isused, the medicament can be easily retained in the inside of the lipidmembrane structures and the like.

As other method for producing a mixture of lipid membrane structures anda medicament in a form of a dispersion in an aqueous solvent, a methodwell known as that for producing liposomes, e.g., the reverse phaseevaporation method or the like, may be separately used. If it is desiredto control the size (particle diameter), extrusion (extrusionfiltration) can be performed under a high pressure by using a membranefilter having uniform pore sizes. Further, examples of the method forfurther drying a dispersion, in which the aforementioned mixture oflipid membrane structures and a medicament is dispersed in an aqueoussolvent, include lyophilization and spray drying. As the aqueous solventin this process, it is preferable to use an aqueous solution of asaccharide, preferably an aqueous solution of sucrose or an aqueoussolution of lactose. Examples of the method for further freezing adispersion, in which the aforementioned mixture of lipid membranestructures and a medicament is dispersed in an aqueous solvent, includeordinary freezing methods. As the aqueous solvent in this process, it ispreferable to use an aqueous solution of saccharide or aqueous solutionof polyhydric alcohol in the same manner as the solution for the lipidmembrane structures alone.

Lipids that can be added to the pharmaceutical composition may besuitably chosen depending on a type of a medicament to be used and thelike. The lipids are used in an amount of, for example, 0.1 to 1000parts by weight, preferably 0.5 to 200 parts by weight, based on 1 partby weight of a medicament when the medicament is not a gene. When themedicament is a gene, the amount is preferably 1 to 500 nmol, morepreferably 10 to 200 nmol, with 1 μg of a medicament (gene).

The method for use of the compounds and the pharmaceutical compositionof the present invention may be suitably considered depending on a modeof usage. The administration route for humans is not particularlylimited, and either oral administration or parenteral administration maybe used. Examples of dosage forms for oral administration include, forexample, tablets, powders, granules, syrups, capsules, solutions forinternal use and the like, and examples of dosage forms for parenteraladministration Include, for example, injections, drip infusion, eyedrops, ointments, suppositories, suspensions, cataplasms, lotions,aerosols, plasters and the like. In the medicinal field, injections anddrip infusion are preferred among them, and as the administrationmethod, intravenous injection, subcutaneous injection and intradermalinjection, as well as local injection to targeted cells or organs arepreferred. Further, as for the cosmetic field, examples of forms ofcosmetics include lotions, creams, toilet water, milky lotions, foams,foundations, lipsticks, packs, skin cleaning agents, shampoos, rinses,conditioners, hair tonics, hair liquids, hair creams and the like.

The administration route of the pharmaceutical composition is notparticularly limited, and either oral administration or parenteraladministration may be used. Parenteral administration is preferred. Theform of the pharmaceutical composition is also not particularly limited.Examples of dosage forms for oral administration include, for example,tablets, powders, granules, syrups and the like, and examples of dosageforms for parenteral administration include, for example, injections,drip infusion, eye drops, ointments, suppositories and the like. Amongthem, injections and drip infusion are preferred, and as theadministration method, intravenous injection as well as local injectionto targeted cells or organs are preferred.

EXAMPLES

The present invention will be explained more specifically with referenceto the following examples. However, the scope of the present inventionis not limited to these examples.

Synthesis Example 1 Synthesis of Polyoxyethylene Pentaerythritol Ether(Average Molecular Weight: 2000)-mono-distearoylphosphadylethanolamineSuccinate

Preparation of Distearoylphosphatidylethanolamine Succinate

Distearoylphosphatidylethanolamine (748 mg, 1 mmol) was added withchloroform (50 mL), stirred at 40° C. and further added withtriethylamine (100 mg, 1 mmol) to obtain a phospholipid chloroformsolution. This solution was added with succinic anhydride (105 mg, 1.05mmol) and reacted at 40° C. for 2 hours. The end point of the reactionwas determined by TLC described below as a point at whichdistearoylphosphatidylethanolamine became no longer detectable byninhydrin coloration. The reaction mixture was cooled and then filteredto remove unreacted distearoylphosphatidylethanolamine. The filtrate wasadded with acetone (100 mL) to obtain crystals ofdistearoylphosphatidylethanolamine succinate (805 mg). Synthesis ofpolyoxyethylene pentaerythritol Ether (average molecular weight:2000)-mono-distearoylphosphadylethanolamine Succinate Preparation ofPolyoxyethylene Pentaerythritol Ether (Average Molecular Weight:2000)-mono-distearoyl-phosphatidyl ethanolamine succinate

Polyoxyethylene pentaerythritol ether (average molecular weight: 2000,m=11, 2.1 g, 1.05 mol) was added with chloroform (20 mL) and dissolved,and then warmed to 40° C. to obtain a uniform solution. Then,distearoylphosphatidylethanolamine succinate obtained in the above (1)was dissolved in chloroform (10 mL), added with triethylamine (100 mg, 1mmol), and added with the solution made uniform by warming to 40° C. Themixture was added with N-hydroxysuccinimide (1.24 g, 0.011 mol) anddicyclohexylcarbodiimide (4.25 g, 0.021 mol) and reacted at roomtemperature for 6 hours. After the reaction, the solvent was removed byusing an evaporator, and the residue was added with toluene (10 mL) andhexane (50 mL) to obtain crystals of polyoxyethylene pentaerythritolether-mono-distearoylphosphadylethanolamine succinate. The crystals wereadded with ethyl acetate (20 mL), dissolved by warming to 40° C., thenadded with hexane (20 mL), stirred and then cooled to a temperaturebelow 10° C. to obtain pentaerythritolpolyoxyethylene-mono-distearoylphosphadyl-ethanolamine succinate (1.8g).

Monitoring of the progress of the reaction and identification of theproduct were performed by thin layer chromatography (TLC) using a silicagel plate. A mixed solvent of chloroform and methanol (volumeratio=85:15) was used as a developing solvent, and the containedsubstance was quantified by coloration with iodine vapor and comparisonwith a standard substance of a known amount. The end point of thereaction was confirmed on the basis of the conversion of the spot ofpolyoxyethylene pentaerythritol ether (average molecular weight: 2000)detected with an Rf value of around 0.6 to 0.7 in TLC described below toa spot detected with an Rf value of around 0.4 to 0.5 due to the bindingwith the phospholipid compound. The product was confirmed on the basisof the change of the peak of the amino group in phosphatidylethanolamine(3000 cm⁻¹) to a peak of carbonyl group (a: 1700 cm⁻¹) in the IRspectrum due to conversion of amino group to amide bond, as well asdetection of succinate (b) at δ: 2.75 ppm (2H, t), 2.95 ppm (2H, t) by¹H-NMR (400 MHz, CDCl₃). Further, existence of phospholipid andoxyethylene chain was confirmed by detection of methyl group at the endof the acyl group of distearoylphosphatidylamine at δ: 0.9 ppm (6H, t),methylene group in the acyl group at δ: around 1.25 ppm andpolyoxyethylene group at δ: around 3.5 ppm in ¹H-NMR.

Synthesis Example 2 Synthesis of Polyoxyethylene Diglycerol Ether(Average Molecular Weight: 5000)-mono-distearoylphosphadylethanolamineglutarate (1) Preparation of DistearoylphosphatidylethanolamineGlutarate

Distearoylphosphatidylethanol (748 mg, 1 mmol) was added with chloroform(50 mL), stirred at 40° C. and further added with triethylamine (100 mg,1 mmol) to obtain a phospholipid chloroform solution. This solution wasadded with glutaric anhydride (138.6 mg, 1.05 mmol) and reacted at 40°C. for 2 hours. The end point of the reaction was determined by TLCdescribed below as a point at which distearoylphosphatidylethanol becameno longer detectable by ninhydrin coloration. The reaction mixture wascooled and then filtered to remove unreacteddistearoylphosphatidylethanolamine, and the filtrate was added withacetone (100 mL) to obtain crystals ofdistearoylphosphatidylethanolamine glutarate (835 mg).

(2) Synthesis of Polyoxyethylene Diglycerol Ether (Average MolecularWeight: 5000)-mono-distearoylphosphadylethanolamine Glutarate

Polyoxyethylene diglycerol ether (average molecular weight: 5000, m=28,5.25 g, 1.05 mmol) was added with chloroform (40 mL), dissolved in itand warmed to 40° C. to obtain a uniform solution. Then,distearoylphosphatidylethanolamine succinate obtained in the same manneras in Synthesis Example 1 was dissolved in chloroform (10 mL) and addedwith triethylamine (100 mg, 1 mmol). The reaction and purification wereperformed in the same manner as in Synthesis Example 1 to obtainpolyoxyethylene diglycerol ether-mono-distearoylphosphadylethanolaminesuccinate (4.0 g).

Monitoring of the progress of the reaction and identification of theproduct were performed by thin layer chromatography (TLC) in the samemanner as in Synthesis Example 1. The end point of the reaction wasconfirmed by TLC described below on the basis of the conversion of thespot of polyoxyethylene diglycerol ether (average molecular weight:5000) detected with an Rf value of around 0.6 to 0.7 to a spot detectedwith an Rf value of around 0.4 to 0.5 due to the binding with thephospholipid compound. The product was confirmed on the basis of thechange of the peak of the amino group in the phosphatidylethanolamine(3000 cm⁻¹) to a peak of carbonyl group (1700 cm⁻¹) in the IR spectrumdue to conversion of amino group to amide bond, as well as detection ofsuccinate (—OCOCH₂CH₂CONH—) at δ: 2.7 ppm (2H, t), 2.9 ppm (2H, t) by¹H-NMR (400 MHz, CDCl₃). Further, existence of phospholipid andoxyethylene chain was confirmed by detection of methyl group at the endof the acyl group in distearoylphosphatidylamine at δ: 0.9 ppm (6H, t),methylene group in the acyl group at δ: around 1.25 ppm andpolyoxyethylene group at δ: around 3.5 ppm by ¹H-NMR.

Synthesis Example 3 Synthesis of Polyoxyethylens Hexaglycerol Ether(Average Molecular Weight: 2000)-mono-distearoylphosphadylethanolaminesuccinate

Polyoxyethylene hexaglycerol ether (average molecular weight: 2000, m=6,2.1 g, 1.05 mol) was added with chloroform (20 mL) and dissolved, andthen warmed to 40° C. to obtain a uniform solution. Then,distearoylphosphatidylethanolamine succinate obtained in the same manneras in Synthesis Example 1 was dissolved in chloroform (10 mL) and addedwith triethylamine (100 mg, 1 mmol). In the same manner as in SynthesisExample 1, polyoxyethylene hexaglycerolether-mono-distearoylphosphadylethanolamine succinate (1.1 g) wasobtained.

Monitoring of the progress of the reaction and identification of theproduct were performed by thin layer chromatography (TLC) in the samemanner as in Synthesis Example 1. The end point of the reaction wasconfirmed by TLC described below on the basis of the conversion of thespot of polyoxyethylene hexaglycerol ether (average molecular weight:2000) detected with an Rf value of around 0.6 to 0.7 to a spot detectedwith an Rf value of around 0.4 to 0.5 due to the binding with thephospholipid compound. The product was confirmed on the basis of thechange of the peak of the amino group in the phosphatidylethanolamine(3000 cm⁻¹) to a peak of carbonyl group (1700 cm⁻¹) in the IR spectrumdue to conversion of amino group to amide bond, as well as detection ofsuccinate (—OCOCH₂CH₂CONH—) at δ: 2.7 ppm (2H, t), 2.9 ppm (2H, t) by¹H-NMR (400 MHz, CDCl₃). Further, existence of phospholipid andoxyethylene chain was confirmed by detection of methyl group at the endof the acyl group in distearoylphosphatidylamine at δ: 0.9 ppm (6H, t),methylene group in the acyl group at δ: around 1.25 ppm andpolyoxyethylene group at δ: around 3.5 ppm by ¹H-NMR.

Synthesis Example 4 Synthesis of Polyoxyethylene Glycerol Ether (AverageMolecular Weight: 2000)-mono-distearoylphosphadylethanolamine Succinate

Polyoxyethylene glycerol ether (average molecular weight: 2000, m=15,2.1 g, 1.05 mol) was added with chloroform (20 mL) and dissolved, andthen warmed to 40° C. to obtain a uniform solution. Then,distearoylphosphatidylethanolamine succinate obtained in the same manneras in Synthesis Example 1 was dissolved in chloroform (10 mL) and addedwith triethylamine (100 mg, 1 mmol), and polyoxyethylene glycerolether-mono-distearoylphosphadylethanolamine succinate (1.9 g) wasobtained in the same manner as in Synthesis Example 1.

Monitoring of the progress of the reaction and identification of theproduct were performed by thin layer chromatography (TLC) in the samemanner as in Synthesis Example 1. The end point of the reaction wasconfirmed by TLC described below on the basis of the conversion of thespot of polyoxyethylene glycerol ether (average molecular weight: 2000)detected with an Rf value of around 0.6 to 0.7 to a spot detected withan Rf value of around 0.4 to 0.5 due to the binding with thephospholipid compound. The product was confirmed on the basis of thechange of the peak of the amino group in the phosphatidylethanolamine(3000 cm⁻¹) to a peak of carbonyl group (1700 cm⁻¹) in the IR spectrumdue to conversion of amino group to amide bond, as well as detection ofsuccinate (—OCOCH₂CH₂CONH—) at δ: 2.7 ppm (2H, t), 2.9 ppm (2H, t) by¹H-NMR (400 MHz, CDCl₃). Further, existence of phospholipid andoxyethylene chain was confirmed by detection of methyl group at the endof the acyl group in distearoylphosphatidylamine at δ: 0.9 ppm (6H, t),methylene group in the acyl group at δ: around 1.25 ppm andpolyoxyethylene group at δ: around 3.5 ppm by ¹H-NMR.

Synthesis Example 5 Synthesis of Polyoxyethylene Pentaerythritol Ether(Molecular Weight: 5000)glutaryl-mono-distearoylphosphatidylethanolamine (1) Synthesis ofPolyoxyethylene Pentaerythritol Ether (Molecular Weight: 5000) Glutarate

Polyoxyethylene pentaerythritol ether (molecular weight: 5000, m=28, 5g, 1 mmol) was added with sodium acetate (3.3 mg, 0.04 mmol) and warmedto 100° C. to obtain a uniform solution. Then, the mixture was addedwith glutaric anhydride (0.11 g, 1.1 mol) and reacted at 110° C. for 8hours. The reaction mixture was cooled and then added with isopropylalcohol (20 mL) to obtain crystals of polyoxyethylene pentaerythritolether (molecular weight: 5000) glutarate. The crystals were added withchloroform (15 mL), dissolved by warming to 40° C., then added withN-hydroxysuccinimide (0.12 g, 1.1 mmol) and dicyclohexylcarbodiimide(0.43 g, 2.1 mmol), and reacted at 40° C. for 2 hours. After thereaction, the reaction mixture was filtered to obtain a solution ofcrude polyoxyethylene pentaerythritol ether (molecular weight: 5000)succinimidylglutarate.

(2) Synthesis of Polyoxyethylene Pentaerythritol Ether (MolecularWeight: 5000) glutaryl-mono-distearoylphosphatidylethanolamine

Distearoylphosphatidylethanolamine (1.5 g, 2 mmol) was added withchloroform (7 mL) and warmed at 40° C. to obtain a phospholipidchloroform solution. Further, this solution was added with triethylamine(0.2 g, 2 mmol) and stirred at 40° C. This phospholipid solution wasslowly added with the aforementioned polyoxyethylene pentaerythritolether (molecular weight: 5000) succinimidyl glutarate solution andreacted at 40° C. for 5 hours. After the reaction, the solvent wasremoved by using an evaporator, and the residue was added with toluene(20 mL), dissolved by warming, then added with hexane (40 mL) andstirred to precipitate crystals. These crystals were separated byfiltration. The resulting crude crystals were added with ethyl acetate(15 mL), dissolved by warming to 50° C., then added with Kyoward 700 andKyoward 1000 (0.1 g for each, Kyowa Chemical Industry Co., Ltd.) asadsorbents and stirred for 80 minutes. Kyowards were removed by suctionfiltration, and the resulting filtrate was added with hexane (10 mL) andcooled to a temperature below 15° C. to precipitate crystals. Thesecrystals were separated by filtration. The resulting crystals were addedwith ethyl acetate (30 mL) and dissolved by warming to 50° C., and thencooled to a temperature below 15° C. to precipitate crystals. Thesecrystals were collected by filtration. When insoluble solids remainedafter the dissolution by warming, they were removed by filtration, andthen the subsequent step was performed. Further, the resulting crystalswere similarly dissolved again in ethyl acetate (20 mL) with warming andadded with hexane (10 mL) to precipitate crystals. The precipitatedcrystals were collected by filtration to obtain crystals with a finalpurity of 98% (5 g, yield: 94.5%).

The end point of the reaction was confirmed by TLC described below onthe basis of the conversion of the spot of polyoxyethylenepentaerythritol ether (average molecular weight: 5000) succinimidylglutarate detected with an Rf value of around 0.7 to 0.8 to a spotdetected with an Rf value of around 0.2 to 0.3 due to the binding withthe phospholipid compound. The product was confirmed on the basis of thechange of the peak of the amino group in phosphatidylethanolamine (3000cm⁻¹) to a peak of carbonyl group (c: 1700 cm⁻¹) in the IR spectrum dueto conversion of amino group to amide bond, as well as detection ofglutarate (d: —OCOCH₂CH₂CH₂CONH—) at δ: 2.0 ppm (8H, m), 2.5 ppm (8H,t), 2.7 ppm (8H, t) by ¹H-NMR (400 MHz, CDCl₃). Further, existence ofphospholipid and oxyethylene chain was confirmed by detection of methylgroup at the end of the acyl group in distearoylphosphatidylamine at δ:0.9 ppm (6H, t), methylene group in the acyl group at δ: around 1.25 ppmand polyoxyethylene group at δ: around 3.5 ppm by ¹H-NMR.

Further, because the hydroxyl value of polyoxyethylene pentaerythritolether used as the raw material was 45 mg KOH/g, and the molecular weightof polyoxyethylene pentaerythritol ether (molecular weight: 5000)succinimidylglutarate was found to be 5812 as measured by gel permeationchromatography (GPC), polyoxyethylene having a molecular weight of about5000 with 4 arms was confirmed. For GPC, SHODEX GPC SYSTEM-11 as asystem, SHODEX RI-71 as a differential refractometer and three columnsof SHODEX KF804L directly connected were used, and tetrahydrofuran wasflowed as a developing solvent at a flow rate of 1 ml/min at a columntemperature 40° C. A 0.1% tetrahydrofuran solution (0.1 ml) of theresulting sample was injected. The molecular weight was calculated byusing BORWIN GPC Calculation Program.

Synthesis Example 6 Synthesis of Polyoxyethylene Hexaglycerin Ether(Molecular Weight: 2000)glutaryl-mono-distearoylphosphatidylethanolamine (1) Synthesis ofPolyoxyethylene Hexaglycerin Ether (Molecular Weight: 2000) glutarate

Polyoxyethylene hexaglycerin ether (molecular weight: 2000, m=6, 2 g, 1mmol) was reacted with glutaric anhydride in the same manner as inSynthesis Example 5 to obtain polyoxyethylene hexaglycerin ether(molecular weight: 2000) glutarate. The reaction product was furtherreacted with N-hydroxysuccinimide (0.12 g, 1.1 mmol) anddicyclohexylcarbodiimide (0.43 g, 2.1 mmol) to obtain crudepolyoxyethylene hexaglycerine ether (molecular weight: 2000)succinimidylglutarate (1.9 g) represented by the following formula (6).

(2) Synthesis of Polyoxyethylene Hexaglycerin Ether (Molecular Weight:2000) glutaryl-mono-distearoylphosphatidylethanolamine

Distearoylphosphatidylethanolamine (1.5 g, 2 mmnol) was reacted withcrude polyoxyethylene hexaglycerine ether (molecular weight: 2000)succinimidylglutarate in the same manner as in Synthesis Example 5, andthe product was further purified to obtain polyoxyethylene hexaglycerinether (molecular weight: 2000)glutaryl-mono-distearoylphosphatidylethanolamine (2.2 g).

The end point of the reaction was confirmed by TLC described below onthe basis of the conversion of the spot of crude polyoxyethylenehexaglycerine ether (average molecular weight: 2000)succinimidylglutarate detected with an Rf value of around 0.7 to 0.8 toa spot detected with an Rf value of around 0.2 to 0.3 due to the bindingwith the phospholipid compound. The product was confirmed on the basisof the change of the peak of the amino group in phosphatidylethanolamine(3000 cm⁻¹) to a peak of carbonyl group (c: 1700 cm⁻¹) in the IRspectrum due to conversion of amino group to amide bond, as well asdetection of glutarate (—OCOCH₂CH₂CH₂CONH—) at δ: 2.0 ppm (8H, m), 2.5ppm (8H, t), 2.7 ppm (8H, t) by ¹H-NMR (400 MHz, CDCl₃). Further,existence of phospholipid and oxyethylene chain was confirmed bydetection of methyl group at the end of the acyl group indistearoylphosphatidylamine at δ: 0.9 ppm (6H, t), methylene group inthe acyl group at δ: around 1.25 ppm and polyoxyethylene group at δ:around 3.5 ppm by ¹H-NMR. Further, because the hydroxyl value ofpolyoxyethylene hexaglycerin ether used as the raw material was 221 mgKOH/g, polyoxyethylene having a molecular weight of about 2000 with 8branches was confirmed.

Synthesis Example 7 Synthesis of Polyoxyethylene Pentaerythritol Ether(Molecular Weight: 2000)succinyl-mono-distearoylphosphatidylethanolamine (1) Synthesis ofPolyoxyethylene Pentaerythritol Ether (Molecular Weight: 2000) Succinate

Polyoxyethylene pentaerythritol ether (molecular weight: 2000, m=11, 2g, 1 mmol) was added with sodium acetate (3.3 mg, 0.04 mmol) and warmedto 100° C. to obtain a uniform solution, then added with succinicanhydride (0.11 g, 1.1 mol) and reacted at 110° C. for 5 hours. Thereaction mixture was cooled and then added with isopropyl alcohol (20mL) to obtain crystals of polyoxyethylene pentaerythritol ether(molecular weight: 2000) succinate. These crystals were added withchloroform (15 mL) and dissolved by warming to 40° C., then added withN-hydroxysuccinimide (0.12 g. 1.1 mmol) and dicyclohexylcarbodiimide(0.43 g, 2.1 mmol) and reacted at 40° C. for 2 hours. After thereaction, the reaction mixture was filtered to obtain a solution ofcrude polyoxyethylene pentaerythritol ether (molecular weight: 2000)succinimidylsuccinate.

(2) Synthesis of Polyoxyethylene Pentaerythritol Ether (MolecularWeight: 2000) succinyl-mono-distearoylphosphatidylethanolamine

Distearoylphosphatidylethanolamine (1.5 g, 2 mmol) was added withchloroform (7 mL) and heated to 40° C. to obtain a phospholipidchloroform solution. Further, the solution was added with triethylamine(0.2 g, 2 mmol) and stirred at 40° C. This phospholipid solution wasslowly added with the aforementioned solution of crude polyoxyethylenepentaerythritol ether (molecular weight: 2000) succinimidylsuccinate andreacted at 40° C. for 5 hours. After the reaction, the solvent wasremoved by using an evaporator, and the residue was added with toluene(20 mL) and dissolved by warming, then added with hexane (40 mL) andstirred to precipitate crystals. These crystals were separated byfiltration. The resulting crude crystals were dissolved in ethyl acetate(15 mL) with warming at 50° C., added with Kyoward 700 and Kyoward 1000(0.1 g for each) as adsorbents and stirred for 30 minutes. Kyowards wereremoved by suction filtration, and the resulting filtrate was added withhexane (10 mL) and cooled to a temperature below 15° C. to precipitatecrystals. These crystals were separated by filtration. The resultingcrystals were added with ethyl acetate (30 mL), dissolved by warming to50° C. and then cooled to a temperature below 15° C. to precipitatecrystals. These crystals were collected by filtration. When insolublesolids remained after the dissolution by warming, they were removed byfiltration, and then the subsequent step was performed. Further, theresulting crystals were similarly dissolved again in ethyl acetate (20mL) with warming and added with hexane (10 mL) to precipitate crystals.The precipitated crystals were collected by filtration to obtaincrystals (5 g) with a final purity of 98% (yield: 94.5%).

The end point of the reaction was confirmed by TLC described below onthe basis of the conversion of the spot of crude polyoxyethylenepentaerythritol ether (average molecular weight: 2000)succinimidylsuccinate detected with an Rf value of around 0.7 to 0.8 toa spot detected with an Rf value of around 0.2 to 0.3 due to the bindingwith the phospholipid compound. The product was confirmed on the basisof the change of the peak of the amino group in phosphatidylethanolamine(3000 cm⁻¹) to a peak of carbonyl group (c: 1700 cm⁻¹) in the IRspectrum due to conversion of amino group to amide bond, as well asdetection of succinate (b) at δ: 2.75 ppm (2H, t), 2.95 ppm (2H, t) by¹H-NMR (400 MHz, CDCl₃). Further, existence of phospholipid andoxyethylene chain was confirmed by detection of methyl group at the endof the acyl group in distearoylphosphatidylamine at δ: 0.9 ppm (6H, t),methylene group in the acyl group at δ: around 1.25 ppm andpolyoxyethylene group at δ: around 3.5 ppm by ¹H-NMR.

Further, because the hydroxyl value of polyoxyethylene pentaerythritolether used as the raw material was 26.7 mg KOH/g, and the molecularweight of polyoxyethylene pentaerythritol ether (molecular weight: 2000)succinimidylsuccinate was found to be 2082 as measured by gel permeationchromatography (GPC), polyoxyethylene having a molecular weight of about2000 with 4 branches was confirmed. For GPC, SHODEX GPC SYSTEM-11 as asystem, SHODEX RI-71 as a differential refractometer and three columnsof SHODEX KF804L directly connected were used, and tetrahydrofuran wasflowed as a developing solvent at a flow rate of 1 ml/min at a columntemperature 40° C. A 0.1 weight % tetrahydrofuran solution (0.1 ml) ofthe resulting sample was injected. The molecular weight was calculatedby using BORWIN GPC Calculation Program.

Example 1 Preparation of Lotion (Evaluation as Solubilizer)

Lotion was prepared by using polyoxyethylene pentaerythritol ether(molecular weight: 5000)glutaryl-mono-distearoylphosphatidylethanolamine obtained in SynthesisExample 5. Among the base materials in the composition shown in Table 1,glycerin and propylene glycol were added to purified water and uniformlydissolved. The other base materials were added to ethanol to obtain auniform solution, and then added to the aforementioned purified aqueousphase with stirring and solubilized to obtain lotion.

Composition example: Propylene glycol  5.0% by weight Glycerin  2.0% byweight Oleyl alcohol  0.5% by weight Hydrogenated soybean lecithin  0.5%by weight Ethanol  7.0% by weight Polyoxyethylene pentaerythritol ether 2.0% by weight (molecular weight: 5000)glutaryl-mono-distearoylphoaphatidylethanolamine Tocopherol 0.02% byweight Fragrant Suitable amount Preservative Suitable amount Purifiedwater 73.0% by weight

Example 2 Preparation of Milky Lotion (Evaluation as Dispersing Agentfor Cosmetics)

Preparation Method of Liposomes

Hydrogenated soybean phosphatidylcholine (645 mg), cholesterol (299 mg)and myristic acid (23 mg) (molar ratio: 1:1:0.1) were added withpolyoxyethylene pentaerythritol ether (molecular weight: 5000) glutarateat a mixed lipid concentration of 5 mole %, added with physiologicalsaline (10 to 11 mL) warmed to 60° C. beforehand at a mixed lipidconcentration of 10% by weight, stirred and further blended in ahomogenizer on a water bath at 60° C. for 10 minutes to obtain aliposome solution.

Among the base materials in the composition shown in Table 2, those ofthe oil phase including an emulsifier were uniformly dissolved by usingthe above liposome solution with warming at 60° C. to form a uniformsolution, and added with the aqueous phase with stirring at the sametemperature to obtain a milky lotion.

Oil phase: Cetanol  2.0% by weight Vaseline  2.0% by weight Squalane 5.0% by weight Liquid paraffin 10.0% by weight Polyoxyethylenemonooleate  2.0% by weight Tocopherol 0.02% by weight Fragrant Suitableamount Preservative Suitable amount Aqueous phase: Propylene glycol 2.0% by weight Purified water 87.0% by weight Liposome solution 10.0%by weight

Comparative Synthesis Example 1 (1) Synthesis ofmonomethylpolyoxyethylenecarbamyl (Molecular Weight: 2000)distearoylphosphatidylethanolamine

Monomethoxypolyoxyethylene (molecular weight: 2000, 20 g, 10 mmol) wasadded with toluene (80 mL), heated to 110° C. and refluxed fordehydration. The residue was added with 1,1′-carbonyldiimidazole (1.95g, 12 mmol) and reacted at 40° C. for 2 hours. The reaction mixture wasadded with pyridine (1.58 g, 20 mmol) anddistearoylphosphatidylethanolamine (7 g, 9.36 mmol) and reacted at 65°C. for 5 hours. The reaction mixture was added with hexane (300 mL) andcrystallized. The crystals were added with ethyl acetate (400 mL),dissolved at 65° C., stirred for 30 minutes, and then cooled to 5° C.The precipitated crystals were collected by filtration. In the samemanner, the step using ethyl acetate was performed once.

The crystals were dissolved in ethyl acetate (400 mL), added withKyoward 700 (1 g) as an adsorbent and stirred at 65° C. for one hour.The reaction mixture was filtered and then cooled to 5° C. forcrystallization. The crystals were washed with hexane (200 mL),collected by filtration and dried to obtainmonomethylpolyoxy-ethylenecarbamyl (molecular weight: 2000)distearoylphosphatidylethanolamine (15.3 g) with a purity of 98.3%(yield: 54.7%).

The product was analyzed by thin layer chromatography (TLC) using asilica gel plate. A mixed solvent of chloroform and methanol with amixing ratio of 85:15 (weight ratio) was used as a developing solvent,and identification and quantification of the contained substance wereperformed by coloration with iodine vapor and comparison with a standardsubstance of a known amount.

Example 3 Determination of Salt Tolerant Effect (Evaluation asSurfactant)

Clouding point of the polyoxyethylene pentaerythritol ether (averagemolecular weight: 2000)-mono-distearoylphosphadylethanolamine obtainedin Synthesis Example 1 was measured for a 1 weight % solution in 5weight % aqueous sodium sulfate. As a result of the measurement, theclouding point was not detectable even when the temperature was raisedup to 80° C.

Comparative Example 1 Comparison of Salt Tolerant Effect (Evaluation asSurfactant)

Clouding point of the monomethylpolyoxyethylenecarbamyl (molecularweight: 2000) distearoylphosphatidylethanolamine obtained in ComparativeSynthesis Example 1 was measured in the same manner as in Example 3. Asa result of the measurement, the clouding point was found to be 50.0° C.This result showed that a branched polyoxyethylene phospholipidderivative exhibited high salt tolerance.

Example 4 Evaluation as Long Circulating Liposomes (1) Preparation ofLiposomes

Lipids in the membrane composition ratios shown in Table 1 (FormulationExamples 1 to 4, Control Examples 1 and 2) were weighed and dissolved ina mixture of chloroform and methanol (2:1). Then, the organic solventwas evaporated by using an evaporator, and the residue was further driedfor one hour under reduced pressure. Then, this dried lipid product(lipid film) was added with 155 mM aqueous ammonium sulfate (pH 5.5, 10ml) warmed to 65° C. beforehand and gently stirred by using a vortexmixer on a warm water bath (until lipids were substantially exfoliatedfrom an eggplant-shaped flask). This lipid dispersion was transferred toa homogenizer and homogenized for 10 strokes, and then the sizingprocedure was performed by using polycarbonate membrane filters withvarious pore sizes (0.2 μm×3 times, 0.1 μm×3 times, 0.05 μm×3 times and0.03 μm×3 times) to prepare an empty liposome dispersion having aparticle diameter of about 100 nm.

This empty liposome dispersion (4 ml) was diluted 2.5 times withphysiological saline. This diluted liposome dispersion was put into aultracentrifugation tube, centrifuged at 65000 rpm for one hour, andthen the supernatant was discarded. The residue was resuspended inphysiological saline in the volume of the liposome dispersion before thecentrifugation (10 ml, the total lipid concentration was adjusted to 50mM at this point). The aforementioned empty liposome dispersion (totallipid concentration: 50 mM) of which external aqueous phase was replacedwith physiological saline and a doxorubicin solution (medicamentconcentration: 3.3 mg/ml of physiological saline) were warmed to 60° C.beforehand, and 4 parts by volume of the empty liposome dispersion wasadded with 6 parts by volume of the doxorubicin solution (that is, thefinal medicament concentration was 2.0 mg/ml, and the final lipidconcentration was 20 mM) and incubated at 60° C. for one hour. Then, themixture was cooled to room temperature to obtain adoxorubicin-containing liposome dispersion.

(2) Physicochemical Properties of Liposomes

The encapsulation efficiency of doxorubicin in the liposomes wasobtained by subjecting a part of the aforementioned liposome dispersionto gel filtration (Sephadex G-50, the mobile phase consisted ofphysiological saline) and quantifying doxorubicin in the liposomefraction eluted in the void volume using liquid chromatography. Further,the particle diameter was measured by the quasi-elastic light scattering(QELS) method using a part of the aforementioned liposome dispersion, Asa result, the encapsulation efficiency of the active ingredient,doxorubicin, in the liposomes was 100% except for Formulation Example 4as shown in Table 1. Therefore, each original liposome dispersion wasused as it was, and diluted 4/3 times with physiological saline for theexperiment for blood retention property in rats described below (thefinal medicament concentration was 1.5 mg/ml, and the final lipidconcentration was 15 mM). Further, the liposomes of Formulation Example4 were subjected to ultracentrifugation (65,000 rpm, one hour) to removethe unencapsulated medicament in the supernatant and adjusted to a finalmedicament concentration of 1.5 mg/ml with physiological saline (thefinal lipid concentration was about 20.0 mM). The particle diameter wasabout 100 nm for all of the liposomes.

(3) Experiment for Blood Retention Property in Rats

An experiment for examining blood retention property was performed in SDmale rats (6-week old) by using the liposome dispersions of FormulationExamples 1 to 4 and Control Examples 1 and 2. Each liposome dispersionwas administered from the cervical veins of rats under etherization(each group consisted of 5 rats, the dose was 7.5 mg doxorubicin/5ml/kg). Then, heparinized blood (0.5 to 1 ml) was collected from thecervical veins under etherization at each of blood collection timepoints (2, 4, 8, 24, 48, 72, 120, 168 hours), and plasma was separated.Then, each sample was pretreated by a conventional method, and plasmaconcentration of the medicament was measured by HPLC. The AUC (0-∞) wasobtained from the concentration of the medicament in plasma in eachliposome dispersion formulation according to the trapezoidal rule. Asshown in Table 1, in comparison with the AUCs of the liposomes ofControl Example 1, which did not contain the lipid derivative of thepresent invention, or the liposomes of Control Example 2, whichcontained only ea phospholipid portion of the lipid derivative of thepresent invention (DSPE: distearoylphosphatidylethanolamine), theliposome formulations containing the lipid derivatives of the presentinvention (Formulation Examples 1 to 4) achieved AUCs greater by one ormore orders, and thus showed evident blood retention property.

TABLE 1 Particle Retention ratio of AUC_(0-∞) ± S.D. Liposome membranecomposition diameter (nm) active ingredient (%) (μg · hr/mL) FormulationDSPE-GLY20H/HSPC/Cholesterol = 88 100.0 3185 ± 662 Example 1 1.04mM/11.28 mM/7.68 mM *DSPE-GLY20H: Synthesized in Synthesis Example 4HSPC: Hydrogenated soybean phosphatidylcholine FormulationDSPE-PTE20H/HSPC/Cholesterol = 108 100.0 6584 ± 739 Example 2 1.04mM/11.28 mM/7.68 mM *DSPE-PTE20H: Synthesized in Synthesis Example 1HSPC: Hydrogenated soybean phosphatidylcholine FormulationDSPE-HGEO20H/HSPC/Cholesterol = 101 100.0 6028 ± 689 Example 3 1.04mM/11.28 mM/7.68 mM *DSPE-HGEO20H: Synthesized in Synthesis Example 3HSPC: Hydrogenated soybean phosphatidylcholine FormulationDSPE-PTESA20H/HSPC/Cholesterol = 68 74.8 4300 ± 494 Example 4 1.04mM/11.28 mM/7.68 mM *DSPE-PTESA20H: synthesized in Synthesis Example 7HSPC: Hydrogenated soybean phosphatidylcholine Control HSPC/Cholesterol= 91 100.0 452 ± 98 Example 1 11.90 mM/8.10 mM *HSPC: Hydrogenatedsoybean phosphatidylcholine Control DSPE/HSPC/Cholesterol = 94 100.0 397 ± 133 Example 2 1.04 mM/11.28 mM/7.68 mM *DSPE:Distearoylphosphatidylcholine HSPC: Hydrogenated soybeanphosphatidylcholine

INDUSTRIAL APPLICABILITY

The phospholipid derivatives of the present invention are highly safefor living bodies, and are useful as surfactants, solubilizers, ordispersing agents in the field of cosmetics and the like. When thephospholipid derivatives of the present invention, which arepolyoxyethylene derivatives having multi-arm, are used for manufactureof lipid membrane structures such as liposomes, the lipid membranestructures are not instabilized, the aggregation of microparticles in anaqueous medium is prevented, and thus a stable state of a solution canbe obtained. Furthermore, liposomes containing the phospholipidderivatives of the present invention are characterized by superior bloodretention property.

1. A phospholipid derivative represented by the following generalformula (1):

wherein Z represents a residue of a compound having 3 to 10 hydroxylgroups; AO represents an oxyalkylene group having 2 to 4 carbon atoms;R¹CO and R²CO independently represent an acyl group having 8 to 22carbon atoms; X represents hydrogen atom, an alkali metal atom, ammoniumor an organic ammonium; “a” represents an integer of 0 to 4; “b”represents 0 or 1; Q represents hydrogen atom or methyl group; mrepresents an average number of moles of the oxyalkylene group added;and m, k1, k2, and k3 are numbers satisfying the following conditions:3≦m≦200, 9≦m×(k1+k2+k3)≦1000, 1≦k1≦2, 0≦k2≦9 and 0≦k3≦9, and3≦k1+k2+k3≦10.
 2. The phospholipid derivative according to claim 1,wherein the condition 4≦k1+k2+k3≦8 is satisfied.
 3. The phospholipidderivative according to claim 1, wherein R¹CO and R²CO independentlyrepresent an acyl group having 12 to 20 carbon atoms.
 4. Thephospholipid derivative according to claim 1, wherein k2 is
 0. 5. Thephospholipid derivative according to claim 4, wherein “a” and “b”represent
 0. 6. The phospholipid derivative according to claim 1,wherein the following conditions k3<1 and k2>k3 are satisfied.
 7. Asurfactant containing the phospholipid derivative according to claim 1.8. A lipid membrane structure containing the phospholipid derivativeaccording to claim
 1. 9. A liposome containing the phospholipidderivative according to claim
 1. 10. A method for producing thephospholipid derivative according to claim 1, which comprises the stepof reacting a phospholipid compound represented by the following generalformula (2):

wherein R¹CO and R²CO independently represent an acyl group having 8 to22 carbon atoms; X represents hydrogen atom, an alkali metal atom,ammonium or an organic ammonium; “a” represents an integer of 0 to 4;and Y represents hydrogen atom or N-hydroxysuccinimide, with apolyalkylene oxide compound represented by the following general formula(3)Z-[—O(AO)_(m)—H]_(k)  (3) wherein Z represents a residue of a compoundhaving 3 to 10 hydroxyl groups; AO represents one or two or more kindsof oxyalkylene groups having 2 to 4 carbon atoms, and when AO representstwo or more kinds of oxyalkylene groups, they may bond to form a blockor random copolymer; m represents an average number of moles of theoxyalkylene group added; and m and k are numbers satisfying thefollowing conditions: 3≦m≦200, 9≦m×k≦1000, and 3≦k≦10, in organicsolvents in the presence of a basic catalyst.
 11. A method for producingthe phospholipid derivative according to claim 1, which comprises thestep of reacting a polyalkylene oxide derivative represented by thefollowing general formula (4):

wherein Z represents a residue of a compound having 3 to 10 hydroxylgroups, “a” represents an integer of 0 to 4; “b” represents 0 or 1; mrepresents an average number of moles of the oxyalkylene group added; Yrepresents hydrogen atom or N-hydroxysuccinimide; and k4 and k5 arenumbers satisfying the following conditions:1≦k4≦10, 0≦k5≦9, and 3≦k4+k5≦10, with a phospholipid derivativerepresented by the following general formula (5)

wherein R¹CO and R²CO have the same meanings as defined in theaforementioned formula (1), in an organi solvent in the presence of abasic catalyst.
 12. A pharmaceutical composition containing the lipidmembrane structure according to claim 8 and a medicament.
 13. Thepharmaceutical composition according to claim 12, wherein the medicamentis an antitumor agent.